It is well known that retroviruses pose a threat to human health by infection of somatic cells, but retroviruses have also been infecting our mammalian ancestors for millions of years and have been incorporated in the germ-line as endogenous retroviruses (ERVs) that account for nearly 10% of our genomic DNA. We are interested in studying ERVs from two perspectives: 1) as a parasite that must be kept in check by the host to prevent widespread viral activation and genomic instability and 2) as a symbiont, that can be co-opted and utilized by the host for evolutionary advantage. Specifically, our work aims to study how the host has adapted molecular recognition machinery to establish stable epigenetic silencing of ERVs in early development following epigenetic reprogramming, how ERVs have sometimes evaded these silencing mechanisms during development, and how these evasive activities have lead to host co-option of viral regulatory sequences that may have contributed to evolution of mammals. Kruppel associated box zinc finger proteins (KRAB-ZFPs) have emerged as candidate transcription factors that serve to recognize and silence ERVs and other mobile parasitic retroelements. KRAB-ZFPs are rapidly evolving transcriptional repressors that emerged in tetrapods and make up the single largest family of transcription factors in mammals (estimated to be greater than 300 in mice and humans) 1. Each species has its own unique repertoire of KRAB-ZFPs, with a small number being shared with other closely related species and a larger fraction specific to each species. Despite their abundance, strikingly little is known about the target genes or physiological functions of the majority of KRAB-ZFPs. KRAB-ZFPs consist of an N-terminal KRAB domain that binds the co-repressor protein KAP1/TRIM282 and a variable number of C-terminal zinc finger domains that mediate sequence specific DNA binding (with each finger recognizing 3 nucleotides of DNA sequence). A trimer of KAP1 interacts with the KRAB domain3, which assembles a number of corepressor proteins, including the histone methyltransferase SETDB1 and heterochromatin protein 1 to initiate heterochromatic silencing4,5. Several lines of evidence point to a potential function of the KRAB-ZFP family in binding and silencing of ERVs. First, the number of C2H2 zinc finger genes in mammals tightly correlates with the number of ERVs 6. Second, the mouse KRAB-ZFP family member ZFP809 was isolated based on its ability to bind to the primer binding site for proline tRNA (PBSPro) of murine leukemia virus (MuLV) 7. Third, we have demonstrated via ChIP-seq and mouse knockout analysis that ZFP809 binds and recruits silencing machinery to a large number of ERVs and LTR transposons in the genome that utilize the PBS-Pro8. Fourth, deletion of the KRAB-ZFP co-repressor Kap1/Trim28 or the histone methylatransferase Setdb1 (which catalyzes the trimethylation of histone H3 lysine 9, H3K9Me3 and associates with KAP1) leads to transcriptional activation of many families of ERVs in mouse ES cells9,10. Thus we have begun a systematic interrogation of KRAB-ZFP function to explore the potential role of this protein family as an adaptive repression system for ERVs. ZFP809 was an obvious choice to begin our interrogation of the function of KRAB-ZFPs that may play a role in ERV silencing. Stephen Goffs group had previously shown that ZFP809 was part of a repression complex that mediated the recognition of exogenously introduced MuLVs via direct binding to the 18 nt Primer Binding site for Proline (PBSpro) sequence7,11. We thus hypothesized that ZFP809 might function in vivo to repress other ERVs and LTR retroelements that utilized the PBSpro. Using ChIP-seq of epitope tagged ZFP809 in embryonic stem (ES) cells and embryonic carcinoma (EC) cells, we determined that ZFP809 indeed bound specifically to several sub-classes of ERV elements via the PBSpro sequence (Fig. 1a). We then generated Zfp809 knockout mice to determine whether ZFP809 was required for silencing these elements. We found that Zfp809 adult knockout tissues displayed high levels of VL30pro elements (Fig. 1b), and that the targeted elements display an epigenetic shift from repressive epigenetic marks (H3K9me3, and CpG methylation) to active marks (H3K9Ac, CpG hypomethylation). ZFP809 mediated repression was also extended to a handful of genes that contained adjacent or internal VL30pro integrations. Furthermore, using a combination of conditional alleles and rescue experiments, we determined that ZFP809 activity was required in ESCs (and preimplantaion embryos) to initiate silencing, but not in somatic cells to maintain silencing of VL30Pro elements. Finally we demonstrated that ZFP809-like genes appeared in Muroidea before the ERVs presently targeted by ZFP809 in the mouse, suggesting that ZFP809 binding preference for PBSpro was selected for by previous PBSpro containing retroviruses. These studies provided the first demonstration for the in vivo requirement for a KRAB-ZFP in the recognition and silencing of ERV elements. They also provided and interesting case study for the adaptive evolution of KRAB-ZFPs against mobile elements. 1 Urrutia, R. KRAB-containing zinc-finger repressor proteins. Genome Biol 4, 231, doi:10.1186/gb-2003-4-10-231 (2003). 2 Friedman, J. R. et al. KAP-1, a novel corepressor for the highly conserved KRAB repression domain. Genes Dev 10, 2067-2078 (1996). 3 Peng, H. et al. Biochemical analysis of the Kruppel-associated box (KRAB) transcriptional repression domain. J Biol Chem 275, 18000-18010, doi:10.1074/jbc.M001499200 (2000). 4 Ryan, R. F. et al. KAP-1 corepressor protein interacts and colocalizes with heterochromatic and euchromatic HP1 proteins: a potential role for Kruppel-associated box-zinc finger proteins in heterochromatin-mediated gene silencing. Mol Cell Biol 19, 4366-4378 (1999). 5 Schultz, D. C., Ayyanathan, K., Negorev, D., Maul, G. G. & Rauscher, F. J., 3rd. SETDB1: a novel KAP-1-associated histone H3, lysine 9-specific methyltransferase that contributes to HP1-mediated silencing of euchromatic genes by KRAB zinc-finger proteins. Genes Dev 16, 919-932, doi:10.1101/gad.973302 (2002). 6 Thomas, J. H. & Schneider, S. Coevolution of retroelements and tandem zinc finger genes. Genome Res 21, 1800-1812, doi:10.1101/gr.121749.111 (2011). 7 Wolf, D. & Goff, S. P. Embryonic stem cells use ZFP809 to silence retroviral DNAs. Nature 458, 1201-1204, doi:10.1038/nature07844 (2009). 8 Wolf, G. et al. The KRAB zinc finger protein ZFP809 is required to initiate epigenetic silencing of endogenous retroviruses. Genes Dev 29, 538-554, doi:10.1101/gad.252767.114 (2015). 9 Rowe, H. M. et al. KAP1 controls endogenous retroviruses in embryonic stem cells. Nature 463, 237-240, doi:10.1038/nature08674 (2010). 10 Matsui, T. et al. Proviral silencing in embryonic stem cells requires the histone methyltransferase ESET. Nature 464, 927-931, doi:10.1038/nature08858 (2010). 11 Wolf, D. & Goff, S. P. TRIM28 mediates primer binding site-targeted silencing of murine leukemia virus in embryonic cells. Cell 131, 46-57, doi:10.1016/j.cell.2007.07.026 (2007).